We have identified, cloned and sequenced three tuf-like genes from Streptomyces ramocissimus (Sr.), the producer of the antibiotic kirromycin which inhibits protein synthesis by binding the polypeptide chain elongation factor EF-Tu. The tuf-1 gene encodes a protein with 71% amino acid residues identical to the well characterized elongation factor Tu of Escherichia coli (Ec.EF-Tu). The genetic location of tuf-1 downstream of a fus homologue and the in vitro activity of Sr.EF-Tu1 show that tuf-1 encodes a genuine EF-Tu. The putative Sr.EF-Tu2 and Sr.EF-Tu3 proteins are 69% and 63% identical to Ec.EF-Tu. Homologues of tuf-1 and tuf-3 were detected in all five Streptomyces strains investigated, but tuf-2 was found in S. ramocissimus only. The three tuf genes were expressed in E. coli and used to produce polyclonal antibodies. Western blot analysis showed that Sr.EF-Tu1 was present at all times under kirromycin production conditions in submerged and surface-grown cultures of S. ramocissimus and in germinating spores. The expression of tuf-2 and tuf-3 was, however, below the detection level. Surprisingly, Sr.EF-Tu1 was kirromycin sensitive, which excludes the possibility that EF-Tu is involved in the kirromycin resistance of S. ramocissimus.